Immunotoxins and antibody drug conjugates (ADCs) are proteinaceous drugs combining a target-specific binding domain with a drug molecule of sufficient potent toxicity that it may be classed as cytotoxic. Antibodies are the ideal biomolecule for this purpose creating a targeting system combining high specificity with high antigen affinity allowing the transportation of the cytotoxic drug direct to the site of desired administration. These drug constructs are potentially therapeutic against diseases, finding particular prevalence within oncology.
The main criteria of an Antibody Drug Conjugate (ADC) are that the toxin ‘warhead’ (drug) has activity at extremely low levels (picoM). Furthermore, it is advantageous to have efficacy towards tumours cells irrespective of the point in the cycle. For this purpose DNA active agents have found favour as toxin candidates as DNA damage, unless repairable, will drive apoptosis irrespective of the point in the cycle.
In principle, a suitable toxin for an ADC can be any moiety defined as a L01 ATC molecule (‘Anatomical Therapeutic Chemical Classification System’ where L01 is a subgroup defining antineoplastic and immunomodulating agents, defined by WHO Collaborating Centre for Drug Statistics Methodology). Alternatively, other moieties that may be categorised as suitable payloads for ADCs may be simply defined as anything that is toxic to cells once internalised. Most moieties falling in the latter category would lack sufficient potency to be effective. Hence, there is an industry trend to identify and exploit ‘ultra-potency’ materials.
An expert review on the rationale, design and effectiveness of immunotoxin and ADC research can be found within: J. Adair et al, Expert Opin. Biol. Ther., 2012, 12(9): P1191-206, G. Casi et al, Journal of Controlled Release, 2012, 161, 2, P 422-428 and F. Dosio et al, Toxins, 2011, 3, P 848-883.
A number of solution-phase methods can be used to manufacture biomolecule-drug-conjugates, e.g. antibody-drug-conjugates (ADCs). However, solution phase methods are themselves wasteful in terms of generating large volumes of waste and are problematic in terms of aggregation of the biomolecule-drug-conjugates during synthesis.
The first step in a solution-phase method for manufacturing biomolecule-drug-conjugates generally involves chemical modification or activation of the biomolecule. For example, where the biomolecule is an antibody, the antibody can be ‘chemically modified’ or ‘activated’ by reducing or partially reducing the antibody. A suitable process for partial reduction of antibodies is given in “Bioconjugate Techniques”, page 96/97, Greg T. Hermanson, Academic Press; 2nd edition, 2008, ISBN-13: 978-0123705013. A reducing agent such as TCEP is generally employed in the reduction process.
After chemical modification or activation of the antibody, e.g. reduction, the next step is to remove any excess activation/chemical modification agent, e.g. excess reducing agent. This step is very time consuming as it is sometimes necessary to run the sample through a separation column multiple times. This can also be problematic in terms of degradation if stability of the biomolecule is an issue. The issue of purification of the chemically modified/activated biomolecule is particularly problematic if the process involves the full reduction of a ThiomAb with a large excess of a reducing agent.
After the above purification step, the chemically modified/activated, e.g. reduced, antibody is then be conjugated with a drug moiety. The major problem with this step is the high probability of aggregation of the biomolecule-drug-conjugate. This is particularly problematic when highly hydrophobic drugs are employed in the process. Aggregation is a major problem as it can lead to unusable biomolecule-drug-conjugates. In the best case scenario, biomolecule-drug-conjugates contaminated with biomolecule-drug-conjugate aggregates must be further purified to remove the aggregates, which is both time consuming and very wasteful. A large proportion of the drug will be lost during purification as it forms part of the aggregated biomolecule-drug-conjugate. In the worst case the entire batch of biomolecule-drug-conjugate contaminated with biomolecule-drug-conjugate aggregate to such a high degree it is entirely unusable and must be disposed of.
Oncologists have been working on harnessing target-specific monoclonal antibodies to deliver cytotoxic drugs to the site of tumors as long as monoclonal antibodies have existed; nearly three decades. Up until now three classes of toxin have dominated the field. Namely, calicheamicins, maytansines and auristatins. These cytotoxic drug classes are all typically hydrophobic in nature. When conjugated to an antibody their presence increases the overall hydrophobicity of the antibody significantly and in some cases to the extent that hydrophobic interactions between conjugates leads to conjugate aggregation. The order of significance of this issue is Calicheamicin>Maytansine>Auristatin based on the knowledge that the processes for both Mylotarg and CMC-544 contain chromatographic aggregate removal steps. Approximately 50% of maytansine processes contain aggregate removal steps and very few auristatin processes contain aggregate removal steps.
More recently, toxins based on duocarmycins (www.syntarga.com) and pyrollebenzodiazepene (PBD) dimers (www.spirogen.com) have been conjugated to antibodies and are undergoing pre-clinical evaluation. These new classes of toxin are even more hydrophobic than their predecessor cytotoxin drug classes and are more prone to aggregation when conjugated to antibodies.
Significant efforts have been focussed on modulation of the hydrophobicity of the drug by incorporating hydrophilic linkers (Zhao et al, J. Med. Chem., 2011, 54, 10, 3606-3623). Where aggregate formation cannot be controlled developers have relied on well-known techniques for aggregate removal from protein based therapeutics. These include a range of different chromatographic separations including ion exchange, hydrophobic interaction, hydroxyapatite and others well known to those in the art. Undertaking such chromatographic purification techniques has the result of achieving adequate product quality but at the expense of process yield. When working with antibodies and antibody based therapeutics in the context of manufacturing physical loss of material through ambiguous, incidental side reactions or unwanted physiochemical interactions has a hugely significant financial impact.
Accordingly, the conventional solution-phase processes for manufacturing biomolecule-drug-conjugate are beset with difficulties and it would be desirable to provide an improved process for manufacturing biomolecule-drug-conjugates.
The present invention addresses one or more of the above issues with the conventional solution-phase methods.